title: Effects of enzymatically inactive recombinant botulinum neurotoxin type A at the mouse neuromuscular junctions.
abstract: Botulinum neurotoxin A (BoNT/A) is used clinically to treat several neurological and metabolic diseases. However, the mechanisms that underlie the clinical use of the toxin remain still to be elusive. BoNT/A inhibits acetylcholine (ACh) release at the motor nerve terminals (MNT) and causes neuroparalysis. The toxic effects of BoNT/A at the MNT occur in sub-pico molar range, and it is invaluable to determine the half-life and the persistence of catalytic activity of the toxin to develop therapeutics against BoNT/A intoxication. However, the use of extremely low concentrations of BoNT/A in cellular, or animal models due to high toxicity makes it difficult to determine new cellular mechanisms and binding or interacting partners of BoNT/A. In order to address this, a catalytically deactivated, non-toxic version of BoNT/A, designated as DrBoNT/A, was characterized. DrBoNT/A lacks endoprotease activity (SNAP-25 cleavage) at concentrations as high as 46,875-fold, compared to wild-type BoNT/A. Unlike BoNT/A injection (3.2 pg), injection of the recombinant product (150 ng or 3.2 pg) into mouse hind limbs failed to cause neuroparalysis as exhibited by the lack of inhibition of toe spread reflex (ability of the mouse to spread its hindlimb toes), and inhibit ACh release at the MNT. The in vitro experiments also demonstrate that DrBoNT/A uptake (at concentrations equivalent to BoNT/A), internalization and localization at the MNT remained unaltered. In addition, modeling studies support that DrBoNT/A lacked the zinc binding ability, and the ability to directly participate in the hydrolysis of SNAP-25 substrate. Collectively, we demonstrate that DrBoNT/A is non-toxic to the MNT and can be used as a surrogate tool to understand the mechanism by which BoNT/A modulates signal transduction mechanisms.
authors.affiliation: Neuroscience Program, School of Pharmacy, College of Health Sciences, University of Wyoming, 1000 East University Avenue, Laramie, WY 82071, USA.
authors.forename: Padmamalini
authors.initials: P
authors.lastname: Baskaran
authors.forename: Teresa E
authors.initials: TE
authors.lastname: Lehmann
authors.forename: Elena
authors.initials: E
authors.lastname: Topchiy
authors.forename: Nagarajan
authors.initials: N
authors.lastname: Thirunavukkarasu
authors.forename: Shuowei
authors.initials: S
authors.lastname: Cai
authors.forename: Bal Ram
authors.initials: BR
authors.lastname: Singh
authors.forename: Sharad
authors.initials: S
authors.lastname: Deshpande
authors.forename: Baskaran
authors.initials: B
authors.lastname: Thyagarajan
mesh_terms: D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D019274:Botulinum Toxins, Type A / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*; D006207:Half-Life; D066298:In Vitro Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology / Q000633:toxicity*
chemical_list: D011994:Recombinant Proteins; D019274:Botulinum Toxins, Type A; D000109:Acetylcholine
keywords: ACh; Acetylcholine release; BoNT/A; Botulinum neurotoxin A; Deactivated; DrBoNT/A; EDL; EPC; HC; HEPES Ringer Solution; HRS; LC; MNT; NMPs; Nerve muscle preparation; Neuromuscular junction; Non-toxic botulinum neurotoxin A; Recombinant botulinum neurotoxin A; TSR; acetylcholine; deactivated recombinant BoNT/A; endplate current; extensor digitorum longus; heavy chain; light chain; mEPC; miniature EPC; motor nerve terminals; nerve muscle preparations; toe spread reflex